|
| Status |
Public on Oct 01, 2010 |
| Title |
stem cell primary culture, rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Total RNA from primary culture
|
| Organism |
Mus musculus |
| Characteristics |
cell line: R1
cell type: mouse embryonic stem cells
|
| Treatment protocol |
Manual dilution method: To investigate nonprinted cells, 0.5 µl samples were drawn from the cell solution and pipetted into the three wells as controls. The controls had 1000 cells on average.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
100 µL of RLT Buffer solution (QIAGEN, 1% mercaptoethanol) was used to lyse the ejected cells and to preserve the RNA. Qiagen RNeasy Minikit (QIAGEN) was used to purify the total RNA.
|
| Label |
Cy5
|
| Label protocol |
2-3 ng sample (total RNA) is primed with the T7 oligo (dT) primer to synthesize cDNA containing a T7 promoter sequence. Second-strand cDNA synthesis converts the single-stranded cDNA into a double-stranded DNA (dsDNA) template for transcription. In vitro transcription to synthesize cRNA generates multiple copies of biotinylated cRNA from the double-stranded cDNA templates.
|
| |
|
| Hybridization protocol |
10 μg of labeled cRNA was fragmented, denatured at 90°C for 5 min, and then hybridized at 37°C for 20 hours using direct hybridization array kits (from AppliedMicroarrays CodeLink). The arrays were then washed in buffer at 47°C for 60 minutes. After washing, the arrays were incubated at room temperature for 30 minutes with a fluorophore (Streptavidin-Alexa Fluor 647, Invitrogen Eugene, OR), run through a series of 5 minute rinses, and spun dry.
|
| Scan protocol |
Arrays were scanned on a GenePix 4000B scanner (Axon Instrument, Sunnyvale, CA).
|
| Description |
2.1C
|
| Data processing |
Raw intensities were normalized by CodeLink software based on global median for each array. The gene expression measurements were further normalized to a median expression level of 1.0 across the arrays. 1000 genes with the highest expression levels “>1” in the experimental samples was used for comparisons.
|
| |
|
| Submission date |
Sep 23, 2010 |
| Last update date |
Sep 25, 2010 |
| Contact name |
Lingsheng Dong |
| Organization name |
Brigham and Women's Hospital
|
| Department |
Renal Division
|
| Street address |
75 Francis Street
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL8063 |
| Series (1) |
| GSE24330 |
Drop-on-demand single cell isolation and total RNA analysis |
|
